Differentiation-dependent gene expression is a hallmark of biological systems/cell types that undergo differentiation. Thus, understanding normal and abnormal keratinocyte differentiation, requires an understanding of the mechanisms that regulate gene expression. This includes identifying ligands that initiate the regulation, understanding events that transfer the regulation from the cell surface to the nucleus, and describe nuclear mechanisms that regulate target genes. We have developed the human involucrin (hINV) gene as an advanced model for the study of differentiation-dependent gene expression. As part of this effort, we have identified transcription factors (junB, junD, Fra-1) that regulate hINV gene expression by binding to specific DNA binding sites within the hINV gene upstream regulatory region. Knowledge of these nuclear events has made it possible to begin examining the signal transduction cascade that transfers the gene regulatory signal from the cell surface to these transcription factors in the nucleus (Effimova et al., J. Biol. Chem. 273:24387, 1998). We initiated this study because in spite of the importance of these signaling cascades in other systems, little is known regarding the steps that transfer regulatory information to the nucleus for keratinocyte genes that are regulated during differentiation. This published study initiates a comprehensive effort to understand signal transduction for a differentiation-regulated gene, and provides the rationale for the experiments outlined in this proposal. Our studies implicate a mitogen-activated protein kinase (MAPK) signaling cascade that involves protein kinase-c, Ras, MEKK1, MEK3/MEK6, p38, and AP1 family members (junB, junD, Fra-1). This cascade is clearly important, as inhibition of these kinases turn off hINV gene expression. These studies are also innovative, as the p38 MAPKs have not been thought to function as regulators of gene expression during differentiation. However, much needs to be learned, and in the studies outlined in this proposal, we will focus on key points in this cascade for which we have limited knowledge. We will (i) identify the PKC isoforms involved in this regulation, (ii) study the role of MEK3 and MEK6 as regulators of p38 activity and determine which p38 isoforms are involved in this regulation, (iii) perform studies designed to determine how p38 regulates AP1 transcription factor distribution, level and activation state, and (iv) evaluate the role of these kinases, in vivo, using DNA ballistics (gene gun) delivery to mouse epidermis. These studies are designed to provide new insights regarding regulation of differentiation-dependent gene expression in epidermis. As involucrin is one of many genes that are regulated during differentiation, we hope and expect that understanding provided by these studies will be useful to other investigators.